Funds are being requested to study the molecular mechanism of respiratory oxidases. Virtually all aerobic organisms on the planet utilize a respiratory oxidase from either the heme-copper superfamily or from the bd-family of enzymes. We are studying both groups of enzymes as part of the proposed research. All of the oxidases reduce dioxygen to water, and use the free energy to generate a voltage across the membrane. Most of the charge separation that is driven by these enzymes comes from protons moving across the membrane within the oxidase protein, either to the enzyme active site, to be consumed to form water, or to the bulk solution on the opposite side of the membrane from which protons are taken up, i.e., proton pumping. We are primarily interested in the pathways through which protons move within the proteins, and in the driving forces that result in proton translocation. Which groups are protonated and when? The catalytic cycle consists of a sequence of proton-coupled electron transfer reactions, and we are interested in how the electron transfer is coupled to moving protons, including the proton pump. We use rapid kinetics techniques to examine individual steps in the reaction, monitoring charge movements across the membrane by time- resolved voltage measurements, and correlating this to the electron transfer events, monitored by UV/vis spectroscopy. FTIR spectroscopy is also utilized to monitor changes in protein structure and the protonation of individual residues. Several X-ray structures serve as guides for site-directed mutagenesis, and we can characterize each mutant by measuring the uptake and release of protons from the enzymes, as well as intra-protein proton translocation. In the next grant period, the emphasis will be on trying to characterize intermediates in the reaction chemistry, and to expand our studies to include "non-canonical" oxidases, which lack what we have assumed to be essential residues, although these "non-canonical oxidases function perfectly well. The human mitochondrial aa3-type cytochrome c oxidase is a member of the heme-copper superfamily. One aspect of medical interest concerns the many mitochondrial genetic defects which influence the function of the mitochondrial oxidase, leading to insufficient ability to make ATP and a variety of symptoms depending on the nature of the lesion. A second point of medical relevance concerns the possible vulnerability of pathogenic bacteria to agents that compromise their aerobic respiration. The need for pathogens to survive under conditions of low oxygen, as in intracellular regions, often requires specialized oxidases, such as the bd-type of cbb3-type enzymes.